CN107828712A - 一种抗酸胁迫重组乳酸菌及其应用 - Google Patents

一种抗酸胁迫重组乳酸菌及其应用 Download PDF

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CN107828712A
CN107828712A CN201711351161.5A CN201711351161A CN107828712A CN 107828712 A CN107828712 A CN 107828712A CN 201711351161 A CN201711351161 A CN 201711351161A CN 107828712 A CN107828712 A CN 107828712A
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CN107828712B (zh
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张娟
陈坚
堵国成
朱政明
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Jiangnan University
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Abstract

本发明公开了一种抗酸胁迫重组乳酸菌及其应用，属于生物工程技术领域。本发明通过在乳酸乳球菌L.lactis NZ9000中过量表达了来源于乳酸乳球菌L.lactis NZ9000的TraG基因，得到了一株酸胁迫抗性能力显著提高的重组乳酸乳球菌L.lactis NZ9000(pNZ8148/TraG)。pH4.0条件下胁迫3h，重组菌株的存活率为对照的61.3倍。本发明还提供了一种提高酸胁迫抗性的方法，该方法具有良好的工业应用价值。

Description

一种抗酸胁迫重组乳酸菌及其应用
技术领域
本发明涉及一种抗酸胁迫重组乳酸菌及其应用，属于生物工程技术领域。
背景技术
当乳酸菌被用作工业化生产之后，在其发酵过程中，随着菌体的代谢生长过程，酸性物质也随之产生并积累，导致细胞面临严重的酸胁迫。为维持发酵生产的稳定性并提高生产效率，工业上通常在发酵的过程中通过添加外源中和剂来维持pH处于稳定的范围。例如通过添加碱性物质(氨水或NaOH)来控制发酵环境的pH值。然而碱性物质的添加往往会导致副产物的积累。而副产物中形成的盐类会再次导致细胞处于高渗的环境，从而造成渗透压胁迫的产生，再次影响菌体的生长与代谢。
在低pH的环境条件下，微生物细胞活性显著降低，从而导致发酵产物的生产效率显著下降。因此提高乳酸菌的酸胁迫耐受性对于其在发酵生产中的应用有着重要的意义。目前提高乳酸菌的酸胁迫耐受性的方法主要有：(1)诱变育种，该方法具有简便、类型繁多等特点，但工作量大、效率低是其主要缺点；(2)生化工程策略，已有报道用过外源添加天冬氨酸以提高乳酸菌的酸胁迫耐受性，但该方法的使用造成了生产成本的增加；(3)代谢工程策略，目前利用代谢工程策略提高乳酸菌环境胁迫的方法主要包括构建新的代谢途径、拓展已有代谢途径和削弱已有代谢途径。上述方法或存在成本问题，或存在成功率低的问题。
发明内容
本发明的目的是提供一种抗酸胁迫元器件以提高乳酸乳球菌酸胁迫抗性。
本发明首先提供了抗酸胁迫元器件，其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一株酸胁迫抗性提高的重组乳酸乳球菌，过量表达转运蛋白TraG。
在本发明的一种实施方式中，所述TraG的氨基酸序列是SEQ ID NO.1所示的序列。
在本发明的一种实施方式中，编码所述TraG的核苷酸序列是如SEQ ID NO.2所示的序列。
在本发明的一种实施方式中，编码所述TraG的核苷酸序列来源于Lactococcuslactis NZ9000。
在本发明的一种实施方式中，所述重组菌的宿主为乳酸乳球菌Lactococcuslactis NZ9000。
本发明解决还提供了一种所述重组菌的构建方法，是将编码SEQ ID NO.1所示氨基酸序列的基因连接到表达质粒上获得重组质粒，再分别转化到宿主菌中获得重组菌。
在本发明的一种实施方式中，所述表达质粒为pNZ8148。
在本发明的一种实施方式中，所述宿主菌是Lactococcus lactis NZ9000。
在本发明的一种实施方式中，所述构建方法具体是：将SEQ ID NO.2所示的核苷酸序列克隆到表达质粒pNZ8148上，获得重组质粒pNZ8148/TraG，再将重组质粒转化到宿主菌Lactococcus lactis NZ9000中，得到重组菌株Lactococcus lactis NZ9000(pNZ8148/TraG)。
本发明还提供一种提高乳酸乳球菌酸胁迫抗性的方法，是在乳酸乳球菌中过量表达转运蛋白TraG。
在本发明的一种实施方式中，所述表达转运蛋白TraG的氨基酸序列是SEQ IDNO.1所示的序列。
在本发明的一种实施方式中，所述方法具体是：将SEQ ID NO.2所示的核苷酸序列克隆到表达质粒pNZ8148上，获得重组质粒pNZ8148/TraG，再将重组质粒转化到宿主菌Lactococcus lactis NZ9000中，得到重组菌株Lactococcus lactis NZ9000(pNZ8148/TraG)，诱导表达TraG。
本发明还提供所述重组乳酸乳球菌在食品、饲料、精细化学品领域的应用。
本发明通过在乳酸乳球菌中过量表达TraG转运蛋白，得到了一株酸胁迫抗性显著提高的重组乳酸菌Lactococcus lactis NZ9000(pNZ8148/TraG)。在酸胁迫条件下，pH4.0胁迫3h后，重组菌株Lactococcus lactis NZ9000(pNZ8148/TraG)的存活率为对照的61.3倍。
附图说明
图1：重组质粒pNZ8148/TraG的结构图；
图2：重组菌株和对照菌株的生长曲线；
图3：pH4.0条件下重组菌株与对照菌株的存活率对比。
具体实施方式
下面结合具体实施例对本发明做更详细的说明。
实施例1重组菌株的构建
从NCBI数据库的L.lactis NZ9000中得到如SEQ ID NO.2所示的TraG的基因序列，并将其克隆到乳酸乳球菌表达质粒pNZ8148上，获得重组质粒pNZ8148/TraG，再将其电转入宿主菌L.lactis NZ9000中，得到重组菌株L.lactis NZ9000(pNZ8148/TraG)。
具体如下：
根据TraG的基因序列设计分别如SEQ ID NO.3、SEQ ID NO.4所示的引物traG-F、traG-R(表1)，以L.lactis NZ9000的基因组为模板PCR扩增，获得SEQ ID NO.2所示的基因片段。将PCR产物和载体pNZ8148分别用Nco Ⅰ和HindⅢ双酶切，酶切产物经纯化后，进行连接。连接产物转化大肠杆菌MC1061(商业化菌株)感受态，氯霉素平板上筛选阳性克隆，经菌落PCR验证和酶切验证，片段大小正确后再进行测序鉴定，最终获得含有正确序列的重组质粒pNZ8148/TraG(重组质粒结构如图1所示)。然后从重组MC1061中提取重组质粒，电转化L.lactis NZ9000感受态细胞，氯霉素平板上筛选阳性克隆，经菌落PCR验证和酶切验证，片段大小正确后，最终获得含有正确重组质粒的菌株L.lactis NZ9000(pNZ8148/TraG)。
电转化条件为：1μL质粒中与40μL的感受态细胞混合，移入预冷的电转杯中，冰上放置10min。电压2000V，电容25μF，电阻200Ω。电击完毕后，立即向电转杯中加入1mL含有20mM MgCl2和2mM CaCl2的GM17培养基(培养基配方：M17培养基+0.5％Glucose)。然后置于30℃静置培养1.5h，涂布于含有氯霉素的GM17平板上，培养36h，挑取转化子验证。
表1引物
实施例2过量表达TraG菌株的生长性能试验
对于考察菌株在过量表达TraG时的生长情况，将菌株L.lactis NZ9000(pNZ8148/TraG)和L.lactis NZ9000(pNZ8148)(对照)接种于加了10μg/mL氯霉素的GM17液体培养基(1mL)中进行活化，放在30℃培养箱中静置培养过夜。再以2％的接种量将种子液转接至新鲜的氯霉素(10μg/mL)GM17液体培养基中，30℃静置培养。每隔2小时取样，测定600nm波长下的OD值。培养至OD6000.4时加入10ng/mL的nisin诱导表达TraG蛋白。以时间为横坐标，OD600值为纵坐标，绘制生长曲线。
结果如图2所示。经生长性能试验分析，重组菌株的生物量和对照菌株无明显差异，说明在L.lactis NZ9000中过量表达TraG蛋白对菌株的生长性能没有影响。
实施例3酸胁迫条件下耐受性试验
对于考察菌株对酸的耐受性分析试验，分别测定了重组菌株和对照菌株在pH4.0条件下的存活率。
具体操作方式如下：将菌株诱导培养6h，离心收集细胞，经0.85％的生理盐水洗涤两次后重悬于等体积的新鲜的pH4.0(乳酸调节)的GM17(含有10μg/mL氯霉素)中，胁迫不同时间。胁迫后的菌悬液洗涤两次后重悬于等体积的生理盐水中，取10μL重悬液，稀释不同梯度点种于GM17氯霉素平板上测定活菌数和存活率。
经耐受性实验分析，在pH4.0的GM17中胁迫3h后，重组菌株L.lactis NZ9000(pNZ8148/TraG)的存活率为对照的61.3倍，说明重组菌株对酸胁迫的耐受性显著提高。说明通过在L.lactis NZ9000中过量表达TraG的方法可以提高乳酸乳球菌酸胁迫抗性。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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Gly Ala Ala Gly Ala Gly Gly Cys Thr Thr Thr Ala Gly Cys Thr
1310 1315 1320
Ala Cys Ala Thr Thr Cys Cys Gly Thr Ala Ala Ala Cys Gly Thr
1325 1330 1335
Gly Ala Gly Ala Thr Gly Thr Cys Ala Thr Thr Cys Ala Thr Gly
1340 1345 1350
Gly Thr Thr Ala Thr Thr Cys Thr Thr Cys Ala Gly Thr Cys Cys
1355 1360 1365
Cys Thr Gly Gly Cys Gly Cys Ala Gly Cys Thr Thr Ala Ala Ala
1370 1375 1380
Ala Ala Ala Ala Thr Gly Thr Ala Thr Ala Ala Ala Ala Ala Thr
1385 1390 1395
Ala Ala Cys Thr Gly Gly Gly Ala Ala Gly Ala Ala Ala Thr Cys
1400 1405 1410
Gly Thr Thr Gly Ala Thr Ala Ala Cys Thr Gly Thr Gly Cys Ala
1415 1420 1425
Ala Gly Cys Cys Thr Thr Ala Thr Thr Thr Ala Cys Thr Thr Gly
1430 1435 1440
Gly Gly Gly Gly Gly Ala Gly Ala Thr Gly Ala Cys Cys Cys Ala
1445 1450 1455
Ala Ala Gly Ala Cys Thr Thr Thr Ala Gly Ala Gly Thr Thr Thr
1460 1465 1470
Thr Thr Ala Thr Cys Thr Ala Ala Ala Cys Gly Thr Thr Thr Ala
1475 1480 1485
Gly Gly Gly Ala Ala Ala Cys Ala Ala Ala Cys Cys Ala Thr Thr
1490 1495 1500
Thr Cys Thr Cys Thr Thr Ala Gly Ala Ala Ala Gly Cys Ala Thr
1505 1510 1515
Ala Gly Cys Ala Thr Thr Thr Cys Thr Ala Ala Ala Gly Gly Thr
1520 1525 1530
Gly Gly Thr Ala Gly Thr Gly Gly Cys Gly Gly Ala Gly Gly Thr
1535 1540 1545
Thr Cys Thr Gly Ala Ala Ala Gly Thr Cys Gly Ala Gly Ala Thr
1550 1555 1560
Ala Ala Ala Ala Cys Thr Gly Gly Cys Cys Gly Thr Gly Ala Cys
1565 1570 1575
Cys Thr Thr Ala Thr Gly Ala Thr Gly Gly Gly Ala Gly Ala Cys
1580 1585 1590
Gly Ala Ala Ala Thr Cys Gly Cys Cys Ala Cys Thr Ala Thr Thr
1595 1600 1605
Gly Ala Cys Gly Gly Cys Gly Ala Thr Gly Ala Gly Thr Gly Cys
1610 1615 1620
Cys Thr Cys Gly Thr Ala Thr Thr Cys Ala Thr Ala Ala Ala Ala
1625 1630 1635
Ala Ala Ala Gly Ala Ala Cys Gly Thr Gly Gly Cys Thr Thr Cys
1640 1645 1650
Cys Thr Thr Gly Ala Cys Cys Gly Cys Ala Ala Ala Thr Ala Thr
1655 1660 1665
Thr Ala Thr Gly Cys Cys Thr Ala Thr Gly Ala Ala Cys Ala Thr
1670 1675 1680
Cys Cys Thr Ala Gly Ala Gly Cys Thr Gly Ala Thr Gly Ala Ala
1685 1690 1695
Thr Thr Gly Gly Cys Thr Ala Gly Ala Ala Ala Thr Cys Cys Ala
1700 1705 1710
Gly Gly Gly Gly Ala Thr Ala Ala Ala Ala Ala Thr Thr Gly Gly
1715 1720 1725
Thr Thr Thr Ala Ala Cys Thr Ala Thr Cys Gly Cys Cys Gly Thr
1730 1735 1740
Thr Ala Cys Ala Thr Gly Ala Ala Thr Gly Ala Ala Gly Ala Ala
1745 1750 1755
Gly Ala Ala Ala Ala Ala Thr Thr Gly Cys Thr Thr Gly Ala Thr
1760 1765 1770
Gly Ala Thr Gly Thr Gly Cys Gly Ala Cys Gly Thr Gly Gly Cys
1775 1780 1785
Gly Cys Thr Ala Cys Thr Ala Thr Thr Thr Thr Ala Ala Ala Thr
1790 1795 1800
Gly Ala Thr Cys Ala Thr Gly Gly Ala Ala Thr Gly Ala Thr Thr
1805 1810 1815
Cys Ala Ala Thr Cys Thr Gly Ala Ala Ala Thr Thr Cys Cys Thr
1820 1825 1830
Gly Cys Thr Thr Ala Ala
1835
<210> 3
<211> 38
<212> DNA
<213> 人工序列
<400> 3
catgccatgg ggatgaaaaa tcaaaatgat aatcaacc 38
<210> 4
<211> 32
<212> DNA
<213> 人工序列
<400> 4
ccaagctttt aagcaggaat ttcagattga at 32

Claims (8)

1.一种重组乳酸菌，其特征在于，过量表达转运蛋白TraG。
2.根据权利要求1所述的一种重组乳酸菌，其特征在于，所述转运蛋白TraG为来源于乳酸乳球菌Lactococcus lactis NZ9000的一种转运蛋白。
3.根据权利要求1或2所述的一种重组乳酸菌，其特征在于，所述转运蛋白TraG的氨基酸序列如SEQ ID NO.1所示。
4.根据权利要求1～3任一所述的一种重组乳酸菌，其特征在于，宿主为乳酸乳球菌Lactococcus lactis NZ9000，表达载体为pNZ8148。
5.权利要求1～4任一所述重组乳酸菌在食品、饲料、精细化学品领域的应用。
6.一种提高乳酸菌酸胁迫抗性的方法，其特征在于，所述方法是在乳酸菌中过量表达转运蛋白TraG。
7.根据权利要求6所述的方法，其特征在于，所述转运蛋白TraG的氨基酸序列为SEQ IDNO.1。
8.一种抗酸胁迫元器件，其特征在于，包括氨基酸序列如SEQ ID NO.1所示的转运蛋白TraG。
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